Many physiological signals (e.g., sensory, hormonal and neurotransmitter signals) are transduced from extacellular to intracellular environments by cell surface receptors. For example, G-protein coupled receptors (GPCRs) (for a review, see Neer, 1995, Cell 80:249-257), tyrosine kinase receptors and tyrosine phosphatase receptors are involved in signal transduction.
As an example, GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an extracellular portion of a GPCR. The intracellular portion of a GPCR interacts with a G-protein to modulate signal transduction from outside to inside a cell. A GPCR is therefore said to be “coupled” to a G-protein. G-proteins are composed of three polypeptide subunits: an α subunit, which binds and hydolyzes GTP, and a dimeric βγ subunit. In the basal, inactive state, the G-protein exists as a heterotrimer of the α and βγ subunits. When the G-protein is inactive, guanosine diphosphate (GDP) is associated with the α subunit of the G-protein. When a GPCR is bound and activated by a ligand, the GPCR binds to the G-protein heterotrimer and decreases the affinity of the Gα subunit for GDP. In its active state, the G submit exchanges GDP for guanine triphosphate (GTP) and active Gα subunit disassociates from both the receptor and the dimeric βγ subunit. The disassociated, active Gα subunit transduces signals to effectors that are “downstream” in the G-protein signaling pathway within the cell. Eventually, the G-protein's endogenous GTPase activity returns active G subunit to its inactive state, in which it is associated with GDP and the dimeric βγ subunit.
Currently available assays of signal transduction are often hampered by low or non-specific signals. Receptor activation pathways can cross talk, leading to a loss in signal specificity. In addition, some receptors when heterologoously expressed may not function in the normal fashion due to the absence of protein(s) integral to signal transduction function. Additionally, assay tools for monitoring protein-protein interactions of signal transduction are few and cumbersome, such as antibody labeling and not well suited for high throughput screening.
Consequently, the inventors provide new methods and assays components for biochemical and cell-based assays using a newly, functionally identified class of therapeutic target, transducisome proteins. Transducisome proteins as described further herein, assembly and organize many types of signal transduction proteins using PDZ domains to permit or enhance signal transduction.